Cholesterol-Induced Conformational Change in the Sphingomyelin Headgroup

Sphingomyelin (SM) and cholesterol (Cho) are the important lipids for the formation of biologically functional membrane domains, lipid rafts. However, the interaction between Cho and the headgroup of SM remains unclear. In this study, we performed solid-state NMR experiments to reveal the Cho effects on the headgroup conformation using ²H-labeled stearoyl-SM (SSM). Deuterated SSMs at the Cα, Cβ, and Cγ positions of a choline moiety were separately prepared and subjected to NMR measurements to determine the quadrupolar splitting of ²H signals in hydrated SSM unitary and SSM/Cho (1:1) bilayers. Using ²H NMR and ¹³C-³¹P REDOR data, the conformation and orientation of the choline moiety were deduced and compared with those derived from molecular dynamics simulations. In SSM unitary bilayers, three torsional angles in the phosphocholine moiety, P-O-Cα-Cβ, were found to be consecutive +gauche(g)/+g/+g or −g/−g/−g. The orientation and conformation of the SSM headgroup were consistent with the results of our molecular dynamics simulations and the previous results on phosphatidylcholines. The quadrupolar coupling at the α methylene group slightly increased in the presence of Cho, and those at the Cβ and Cγ decreased more significantly, thus suggesting that Cho reduced the gauche conformation at the Cα-Cβ torsion. The conformational ensemble in the presence of Cho may enhance the so-called umbrella effect of the SSM headgroup, resulting in the stabilization of Cho near the SM molecules by concealing the hydrophobic Cho core from interfacial water. We also examined the effect of the chiral centers at the sphingosine chain to the headgroup conformation by determining the enantiomeric excess between the diastereomeric +g/+g/+g and −g/−g/−g conformers using (S)-Cα-deuterated and (R)-Cα-deuterated SSMs. Their ²H NMR measurements showed that the chiral centers induced the slight diastereomeric excess in the SM headgroup conformation.